The goal of this project is to discover novel antibiotics by activating the silent biosynthetic operons of Actinobacteria. Whole genome sequencing of many Actinobacteria showed that there are 10-20 times more operons coding for secondary metabolites than known compounds in a given species. This opens an attractive opportunity to access a large untapped source of new antibiotics. Genetic engineering has been used to turn on silent operons in Actinobacteria, leading to production of secondary metabolites. However, the pace at which engineered operons are activated is very low, less than ten metabolites a year are being reported based on this approach. We reasoned that mutagenesis of isolates that do not produce antimicrobials in vitro will relieve silent operons from regulatory constraints, and screening will then identify the producing mutants. Our preliminary data showed that the approach works surprisingly well, turning over half of the inactive organisms into antibiotic producers. The method is scalable, and we recently identified two potentially novel antimicrobials using this approach. In Phase I, we will mutagenize/screen 2,000 inactive strains for antibiotic production, aiming to obtain new, potentially useful antimicrobials. Biological and chemical dereplication will indicate compounds with potential novelty. We will give priority to broad spectrum compounds with activity against difficult to treat gram-negative pathogens. The antimicrobials will be tested for potency, spectrum, specificity of action and cytotoxicity, and th structure of compounds that pass validation will be determined. Finding 2-3 antimicrobials with novel chemistry will serve as proof-of-principle for this approach. These findings will provide a solid basis for a large-scale drug discovery effort in Phase II.